STEM CELLS AND REGENERATION TECHNIQUES AND RESOURCES ARTICLE

An in vitro model of hemogenic endothelium commitment andhematopoietic productionLaurent Yvernogeau1,2,*, Rodolphe Gautier1,2, Hanane Khoury1,2, Sara Menegatti1,2,‡, Melanie Schmidt1,2,Jean-Francois Gilles3 and Thierry Jaffredo1,2,§

ABSTRACTAdult-type hematopoietic stem and progenitor cells are formed duringontogeny from a specialized subset of endothelium, termed thehemogenic endothelium, via an endothelial-to-hematopoietictransition (EHT) that occurs in the embryonic aorta and theassociated arteries. Despite efforts to generate models, little isknown about the mechanisms that drive endothelial cells to thehemogenic fate and about the subsequent molecular control of theEHT. Here, we have designed a stromal line-free controlled culturesystem utilizing the embryonic pre-somitic mesoderm to obtain largenumbers of endothelial cells that subsequently commit intohemogenic endothelium before undergoing EHT. Monitoring theculture for up to 12 days using key molecular markers revealsstepwise commitment into the blood-forming system that isreminiscent of the cellular and molecular changes occurring duringhematopoietic development at the level of the aorta. Long-termsingle-cell imaging allows tracking of the EHT of newly formed bloodcells from the layer of hemogenic endothelial cells. By modifying theculture conditions, it is also possible to modulate the endothelial cellcommitment or the EHT or to produce smooth muscle cells at theexpense of endothelial cells, demonstrating the versatility of the cellculture system. This method will improve our understanding of theprecise cellular changes associated with hemogenic endotheliumcommitment and EHT and, by unfolding these earliest steps of thehematopoietic program, will pave theway for future ex vivo productionof blood cells.

KEY WORDS: Hemogenic endothelium, Hematopoiesis,Endothelium, Aorta, Avian, Quail

INTRODUCTIONThe first hematopoietic stem/progenitor cells (HSPCs) emergeduring the initial phase of embryonic development in the form ofcell aggregates, designated hematopoietic clusters, that areintimately associated with endothelial cells (ECs) in the floor ofthe dorsal aorta and in the umbilical and vitelline arteries (Dzierzakand Speck, 2008; Medvinsky et al., 2011). These hematopoieticclusters were shown to be pivotal in the formation of the adult bloodsystem by providing the first adult-type hematopoietic stem cells.

Aorta-associated hematopoietic cluster formation has beendocumented in a variety of vertebrate embryos and thoroughlycharacterized by immunohistological studies on sections (Jaffredoet al., 2005b) and, more recently, through high-resolution 3Dvisualization techniques (Yokomizo and Dzierzak, 2010). Tracingexperiments in vivo, including live imaging approaches, revealed adevelopmental relationship between ECs and hematopoietic clusters(Jaffredo et al., 1998; de Bruijn et al., 2002; Zovein et al., 2008;Chen et al., 2009; Bertrand et al., 2010; Boisset et al., 2010; Kissaand Herbomel, 2010; Lam et al., 2010). Cluster emergence relies onthe presence of specialized ECs, termed hemogenic endothelialcells, which, upon appropriate signaling (Richard et al., 2013), losetheir endothelial phenotype and acquire hematopoietic traits. Thiscomplex, multi-step developmental process was designated theendothelial-to-hematopoietic transition (EHT) (Kissa andHerbomel, 2010). During EHT, hematopoiesis was shown tooccur de novo at the expense of the hemogenic endotheliumcompartment that is progressively lost (Pouget et al., 2006; Kissaand Herbomel, 2010).

Since the hemogenic endothelium represents a small fractionof the ECs and is found only during the early stages of developmentin the aorta and the associated arteries, a number of effortshave been devoted to design in vitro models that faithfullyrecapitulate hemogenic endothelium commitment and hematopoieticproduction. The most robust and reliable system was obtained withthe use of embryonic stem cells (ESCs). Using appropriate cultureconditions, ESCs undergo differentiation and form cellularstructures containing progenitors, termed blast colony-formingcells, that are able to give rise to colonies comprising both bloodcells and ECs (Choi et al., 1998). Comparing in vitro cultures withthe in vivo situation in the early mouse embryo, the blast colony-forming cell was shown to be the in vitro equivalent of the nascentmesoderm ingressing through the primitive streak (Huber et al.,2004) and was characterized by the expression of the receptortyrosine kinase Flk1 (also known as Kdr and Vegfr2) and the T-boxtranscription factor brachyury (Robertson et al., 2000). Using well-established culture systems, it was possible to drive this earlymesodermal progenitor to differentiate into hemogenic ECs able togive rise to blood cells through EHT and to document severalaspects of blood formation from hemogenic ECs (Eilken et al.,2009; Lancrin et al., 2009), including some aspects of EHT (Ditadiet al., 2015). However, the number of hemogenic ECs generated inESC cultures was reported to be low (1/3000) (Eilken et al., 2009),hampering the establishment of a versatile culture system that couldbe employed to address questions concerning hemogenicendothelium commitment from non-hemogenic ECs and thecellular and molecular changes that occur during EHT.

The pre-somitic mesoderm (PSM), or paraxial mesoderm, givesrise to the somites and contains numerous progenitor cells able todifferentiate into the various lineages produced by the somites. InReceived 22 May 2015; Accepted 21 February 2016

1Sorbonne Universites, UPMC Univ Paris 06, IBPS, UMR 7622, Laboratoire deBiologie du Developpement, Paris 75005, France. 2CNRS, UMR 7622, Inserm U1156, IBPS, Laboratoire de Biologie du Developpement, Paris 75005, France.3Institute of Biology Paris-Seine, Sorbonne Universites, UPMC Univ Paris 06,Cellular Imaging Facility, Paris 75005, France.*Present address: Hubrecht Institute, Uppsalalaan 8, Utrecht 3584 CT,The Netherlands. ‡Present address: Cancer Research UK Manchester Institute,The University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.

§Author for correspondence (thierry.jaffredo@upmc.fr)

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addition to the axial skeleton, skeletal muscles and dermis, the PSMhas also been shown to produce a small cohort of ECs that give riseto vascularization of the body wall and limbs and contribute to theaortic roof (Pardanaud et al., 1996; Pouget et al., 2006; Yvernogeauet al., 2012). Of note, somite-derived ECs, which are not initiallyendowed with hemogenic potential, can be turned into hemogeniccells following exposure to endoderm or to growth factorsmimicking the endoderm (Pardanaud and Dieterlen-Lievre, 1999).Hence, the PSM appears to be a source of naïve mesodermalprogenitors that can be orientated towards various cell types uponexposure to the appropriate signals. Given the accessibility of PSM,we reasoned that it could be used as an in vitro model in which totrigger the differentiation of large numbers of ECs and hemogenicECs able to recapitulate the cellular events occurring in the aorta atthe time of hematopoietic production.Here, we report the development of such an in vitro, feeder-free

culture system that allows the commitment of naïve mesodermalcells en masse into hemogenic ECs that are able to undergo robustand long-lasting hematopoiesis over a period of at least 2 weeks. Theapproach uses pieces of PSM that are submitted to a cocktail ofgrowth factors that drives them to differentiate into ECs. HemogenicECs, characterized by the expression of the transcription factorRUNX1, are specified from cells already expressing endothelialmarkers. Single-cell tracking of hemogenic ECs allows capture ofthe ephemeral flat-to-round cell transition and revealed unexpectedtraits of EHT. Moreover, by modulating the combination of growthfactors it is possible to preclude EC commitment, accelerate EHT or

to promote smooth muscle cell versus EC differentiation. Thedesign of this robust, highly reproducible in vitro model willimprove our understanding of the precise cellular and molecularchanges associated with EHT and will pave the way for the futureex vivo production of blood cells and potentiate the discovery ofblood cell modulators.

RESULTSPSM as a naïve mesodermal tissueSomites are segmental mesoderm derivatives known to be pivotal inthe formation of vertebrate embryos by producing a variety of celltypes, including skeletal muscles, dermis of the back, tendon, axialskeleton and endothelium of the body wall and limbs (Christ et al.,2007). The somites differentiate from the rostral end of the PSM, aband of loose mesenchyme that does not exhibit any segmentalpattern but expresses an ensemble of oscillatory genes to form thesomites (Pourquie, 2003). Taking advantage of previous experiencewith the in vivo manipulation of pieces of mesoderm from earlyembryos (Pardanaud and Dieterlen-Lievre, 1999), we hypothesizedthat this tissue could serve as a source for naïve mesoderm that couldbe orientated towards the endothelial lineage and, more specifically,towards the hemogenic endothelial lineage following exposure tothe appropriate signals.

Quail PSM was isolated as described (Pouget et al., 2006;Yvernogeau et al., 2012), free of surrounding contaminating tissues(Fig. 1A, Movie 1). We chose this species because our previous workwas based on the use of quail PSM (Pouget et al., 2006) and because

Fig. 1. Culture system and molecularidentification of the PSM. (A) The two quailPSMs (red) encompassing a length of aboutten somites were isolated, cut into five or sixpieces each, and placed in 35-mm culturedishes coated with collagen I. Ten PSMswere plated per dish in Opti-MEM in thepresence of 5% fetal calf serum, 1% chickenserum, VEGF, FGF, IGF, EGF,hydrocortisone and ascorbic acid. Cultureswere followed over a period of 12 days andtime-lapse imaged at different time points.(B) qRT-PCR analysis showing the identityof the PSM through the expression ofmesodermal, endothelial andhematopoiesis-specific genes. A positive/negative fold change representsupregulation/downregulation, respectively,of expression in the PSM relative to thesame stage embryo. RNA for qPCR wasobtained by pooling ten PSMs from fiveembryos of the same stage. n=2independent experiments. Error barsindicate s.d.

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quail cells are known to display greater multiplication potential inculture than chicken cells. PSM was submitted to qRT-PCR analysisto probe the expression of mesoderm genes as well as endothelial andhematopoiesis-specific genes (Fig. 1B). Indeed, the PSM expressedbrachyury (BRA), which encodes a T-box molecule that is expressedby epiblast cells and by the nascent mesoderm following gastrulation(Wilkinson et al., 1990; Kispert et al., 1995), and MEOX1, which isspecific for uncommitted mesoderm and trunk paraxial mesoderm,but was free of endothelial and hematopoiesis-specific geneexpression indicating that none of these lineage commitments hasoccurred at the time of PSM isolation.

Design of culture conditions and the observation of culturesWe tested different culture media and extracellular matrices thatcould favor adhesion and the differentiation of PSM-derived cellstowards the endothelial lineage. The pieces of PSM exhibited a poorcapacity to spread in the absence of extracellular matrix (notshown). Since collagen was reported to positively regulate ECcommitment and migration (Whelan and Senger, 2003; Lamaliceet al., 2007), we compared collagen I and IV in their efficacy tosupport endothelial differentiation. Based on cell morphology(Hirashima et al., 2003; Guo et al., 2007; Eilken et al., 2009), type Icollagen-coated dishes offered a better differentiation of PSM cellsthan type IV collagen-coated dishes (not shown). We initially used acocktail of growth factors from Lonza (SingleQuots Kit) reported toimprove the growth of primary EC cultures. However, owing to lackof information regarding the concentrations of the individual growthfactors, we decided to replace this cocktail by one of the samecomposition but using individually purchased growth factors ofknown concentration. Having chosen the medium (Opti-MEM) andthe support (35-mm collagen I-coated dish), the following humangrowth factors and supplements known to work on avian cells (ourunpublished work) were added to Opti-MEM/fetal calf serum (5%)/chicken serum (1%)/penicillin-streptomycin (100 units/ml): VEGF(2 ng/ml), FGF (4 ng/ml), IGF (3 ng/ml), EGF (10 ng/ml),hydrocortisone (200 ng/ml) and ascorbic acid (75 ng/ml).The culture conditions being defined, we then followed

morphological aspects of the culture over a period of 12 days (D).Retrospectively, the culture period could be divided into threephases: (1) spreading and EC differentiation from D0 to D3-4; (2)hematopoietic cell (HC) emergence and production from D4-5 toD8-9; (3) the continuation of HC production and multiplicationfrom D8-9 to D12. During the first 24-48 h of culture (D0 to D2),the pieces of PSM spread onto the collagen I-coated dishes, formingflat layers of tightly adherent cells that displayed EC-likemorphology (Fig. 2A-C, Movie 2). From D4, isolated cellsamong the layer of ECs began to produce round cells thatdetached from the culture (Fig. 2D). Their number increased overthe next few days to form large areas covering the EC layer beneath(Fig. 2E,F). From this period, their number increased further toinvade the whole culture dish within the next 3-4 days (Fig. 2G).Flow cytometry analysis using a monoclonal antibody recognizingthe QH1 antigen (hereafter referred as to QH1), as a marker forendothelial and hematopoietic cells (Pardanaud et al., 1987)indicated that, at D4, ∼70% of the cells were QH1+ (Fig. 2H), asconfirmed by QH1 immunocytological staining of the culture(Fig. 2I,J). The EC phenotype was also confirmed using the uptakeof human AcLDL as readout (Fig. 2K,L).

Molecular characterization of the cultureWe monitored the culture over a period of 12 days and analyzed theexpression of gene sets representative of naïve mesoderm,

endothelium, hemogenic endothelium and hematopoietic cellsusing semi-quantitative RT-PCR (Fig. 3A), with validation ofsome key genes using qPCR (Fig. 3B-D). From D0 to D6, cultureswere analyzed daily, and every 2 days from D6 to D12.

BRA was detected immediately following culture onset andduring D1, but was no longer expressed at D2. Initially absent at theonset of culture, FLK1 expression appeared at D0.5-D1. Co-expression of BRA and FLK1, reported to be associated with thehemangioblast stage (Huber et al., 2004; Vogeli et al., 2006), wasdetected between D0.5 and D1. The transcription factor SCL (alsoknown as TAL1), which is crucial for the emergence of thehematopoietic and endothelial lineages during embryoid bodydifferentiation (Lancrin et al., 2009), was found to be expressedfrom D0.5, as was GATA2, which is known to be expressed bymesoderm and nascent ECs (Elefanty et al., 1997; De Val andBlack, 2009). Expression of the genes encoding the endothelial andhematopoietic progenitor cell antigen CD34 (Tavian et al., 1996;Wood et al., 1997) and, to a weaker extent, the endothelial-specificcalcium-dependent cell adhesion molecule CD144 (also known ascadherin 5) (see also Fig. 3B), was activated at the same time,indicating that endothelial commitment has occurred. This wasaccompanied by the onset of expression of CD31 (also known asPECAM1), which is expressed on endothelial and hematopoieticcells during development (Newman et al., 1990), and of vWF, a keymarker of EC function (Wagner et al., 1982), supporting a dynamicEC commitment. Of note, RUNX1, a key gene in the generation ofblood from the hemogenic endothelium, was expressed at low levelsfrom D0.5 and was significantly upregulated from D3, suggestingthat hemogenic endothelium differentiation occurred shortly afterthe onset of culture. Taken together, ECs differentiated from D0.5,shortly followed by hemogenic ECs.

The culture persisted free of hematopoietic cells until D3. FromD3onwards, the myeloid- and B lymphoid-specific transcriptionalactivator PU1 (also known as PU.1 or SPI1) was expressed,shortly followed by CD45 (also known as PTPRC), a pan-hematopoietic marker during development, indicating thathematopoietic commitment had occurred (see also Fig. 3C,D for amore quantitative analysis). PU1 and CD45 expression weresignificantly reinforced from D5, which is when the production ofround cells first became prominent. From D6, most of the markersmaintained their expression until the end of the culture period at D12.

In order to confirm the commitment into hemogenicendothelium, cultures were co-stained for AcLDL and with anantibody against RUNX1 from D0 to D3. RUNX1 expression wasdetectable by immunocytochemistry by D2. At this time, only a fewAcLDL+ cells expressed RUNX1. The number of RUNX1+ cellsdramatically increased at D3 (Fig. 3E-G, Fig. S1A-C), 1 day beforethe first conspicuous EHT events.

Characterization of the non-adherent cellsWe characterized the non-adherent cell fraction from D4, the onsetof their production, to the end of the culture period at D12, whennon-adherent cells were present in large numbers. Non-adherentcells are hereafter designated as the floating (F) fraction followed bya number indicating their day of retrieval from the culture, i.e. F6refers to non-adherent cells collected on D6. Non-adherent cellswere collected daily from D4 to D12 by thoroughly rinsing theculture dishes, and were counted. A mean of 2-3×104 cells wereproduced daily from F4 to F10. By F10, the production increased toreach 1.04×105 cells by F12 (Fig. 4A). This increase is at least partlydue to the multiplication of the non-adherent cells, as documented inMovie 3.

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Since QH1 marks quail endothelial and hematopoietic cells(Pardanaud et al., 1987), we FACS analyzed the floating fraction forQH1 at D7. More than 98% of the cells were QH1+, indicating thatthey probably exhibit a hematopoietic phenotype (Fig. 4B). FACsanalysis results were confirmed by QH1 immunostaining of thenon-adherent fraction (Fig. 4C,D).With the aim of further characterizing these cells, we

performed qRT-PCR on the non-adherent fraction from D4 toD12 using key markers of endothelial-to-hematopoietic cell

commitment. As expected, BRA expression was never detectedin floating cells (not shown). CD144, a key marker of ECs, wasdownregulated (Fig. 4E) in keeping with the endothelial-to-hematopoietic commitment analyzed in vivo. By contrast, PU1(Fig. 4F) and CD45 (Fig. 4G) were upregulated with time,indicating that a progressive hematopoietic commitment wasoccurring. Interestingly, PU1 expression preceded that of CD45in accordance with a progressive hematopoietic maturation inculture.

Fig. 2. Morphology and characterization of the culture over time. (A-G) Observation of the culture over a period of 12 days. (A) D0.5, showing initial spreadingof a PSM piece. (B) D1, showing the spread of cells to form a flat layer of tightly attached cells, except for the center where spreading is still in progress. (C) D2,showing that cells unable to spread in the center have died. Large areas of adherent cells displaying EC sheet morphology are clearly visible. (D) D5, thebeginning of the budding process. Some round, hematopoietic-like floating cells are visible either as single cells or as aggregates of refracting cells. (E) D7, whenlarge aggregates of refracting cells are visible, with the flat layer of cells displaying EC sheet morphology beneath. (F) D10, the number of round, hematopoietic-like cells has significantly increased to cover large areas of the culture dish. (G) D12, the culture is now covered with round cells. The presence of flat EC-likecells has significantly decreased. Scale bar: 100 µm. (H) Flow cytometry analysis of the culture at D4 with the monoclonal antibody QH1. About 70% of thecells, which are mostly flat, attached cells at that stage, are QH1+, testifying to the prominent presence of endo-hematopoietic cells. (I,J) Immunofluorescentcharacterization of the flat cells using the QH1 monoclonal antibody at D4. Flat cells clearly display cell surface QH1 (J, red), indicative of their endothelialphenotype. (I) Phase contrast. (K,L) AcLDL uptake. Living cells were submitted to AcLDL-A488 uptake for 3 h at 37°C. Endocytic vesicles appear green under UVtransillumination. Most of the cells displayed AcLDL uptake (L, green), indicative of their endothelial phenotype. (K) Phase contrast. Insets show highermagnifications of boxed regions.

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To further identify the non-adherent fraction, we collected F6cells and performed cytospin followed by May–Grünwald Giemsastaining. Thorough characterization of the cells revealed theirhematopoietic phenotype and the presence of cells from thegranulocyte, erythroblast and monocyte lineages (Fig. 4H).

Tracking EHTBased on previous characterization, the production of round cellsfrom flat cells faithfully corresponds to an EHT (Jaffredo et al., 1998;Kissa and Herbomel, 2010). Given the high number of hemogenicECs generated in the culture and the large number of HCs produced,we decided to track EHT using live imaging. Cultures were imagedevery 10-15 min over periods from 14-18 h during D3 to D4, when

the first EHT events are initiated. To better track EHT, we developeda script running under ImageJ that allows: (1) the labeling of a cellundergoing EHT and to retrospectively identify the flat cell givingrise to the round cell; and (2) to follow the bright, newly formedround cell, over time (see Materials and Methods).

EHT was rapid and left a cell-free area indicating that the fatechange occurred at the single-cell level. The passage from flat,adherent cell, to non-adherent cell took between 15 and 30 min. Ingeneral, no cell division was detected prior to the passage from flat toround, ruling out asymmetric cell division as a prerequisite for EHT.Round cell production thus caused a progressive exhaustion of thelayer of flat cells. Upon detachment, the cell underwent dynamicmovements, emitting cellular processes during a period of 30-45 min.

Fig. 3. Molecular characterization of theculture. (A) Semi-quantitative RT-PCRrevealing the sequential expression of sets ofgenes required for mesoderm induction andendothelial and hematopoietic commitment.PCR reactions were run side by side on a 1%agarose gel. Cells from two or three culturedishes were harvested daily from D0 to D6 andevery 2 days from D6 to D12. GAPDH serves asa control for mRNA amount. A progression frommesoderm to hematopoietic commitment isclearly visible through the expression patterns ofseveral lineage-specific genes (see Results fordetails). (B-D) qPCR analysis of endothelial,hematopoietic commitment and hematopoieticdifferentiation-specific genes. The results areshown as the log-transformed fold change inexpression (PSM compared with same stageembryo). (B) CD144, a recognized endothelial-specific gene, displays a transient increase inexpression from D0.5 to D4, then drops andremains low but stable from D6 to D12. (C) PU1,one of the earliest genes expressed duringhematopoietic commitment, becomesexpressed by D3, indicating the onset of EHT. Itsexpression increased with time and remainedhigh at D12. (D) CD45, a hematopoiesis-specificgene, becomes expressed at low level by D3,then is strongly increased by D6 indicative ofsubstantial hematopoietic differentiation andremains high until D12. Error bars indicate s.d.(E-G) Uptake of AcLDL and expression of thetranscription factor RUNX1 in D4 cultures.(E) Phase contrast. The flat endothelial-specificphenotype is clearly visible. (F) Cells displayprominent uptake of AcLDL. (G) Several cellsdisplay high RUNX1 immunostaining in thenucleus, while weaker staining is also visible inmost of the cells indicative of their hemogenicendothelial commitment.

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Following these movements, cells became round and left the site ofproduction. In rare instances, round cells immediately initiated celldivision, whereas the vast majority of round cells that were producedunderwent cell division some hours after EHT (Movies 4-6).

Influence of the tissue of origin and the composition of themediumGiven the results obtained with the PSM, we examined whetherother conditions could give similar or superior EC commitment. Wefirst compared the PSM with the four last-formed somites and withthe lateral plate mesoderm isolated from embryos at the same stage.The pieces of tissue were cultured in the above-described conditionsover a period of 4 days, i.e. to just before the initiation of EHT. ECcommitment was first assessed based on morphological criteriawhen ECs were conspicuously visible, and second using FACS

analysis following AcLDL uptake. As expected, the PSM gave riseto flat layers of tightly adherent cells that displayed EC-likemorphology, whereas the somites and the lateral plate mesodermshowed a poor EC-like differentiation. In addition, cells of the lateralplate did not exhibit robust growth in the culture conditionsemployed (Fig. S2A,C,E) and FACS analysis was therefore notperformed. From three independent experiments the PSM gave amean of 40±13%AcLDL+ cells, whereas the somites gave amean of3±3% (Fig. S2B,D), demonstrating the unique capacity of PSM cellsto respond to EC commitment under our culture conditions. It shouldbe noted that this percentage is slightly lower than that we reportedusing QH1 staining. This might be related to subtle differences in thematuration status of the ECs, i.e. the QH1 antibody recognizes theimmature ECs (angioblasts) and the more mature ECs whereasAcLDL stains the mature ECs.

Fig. 4. Characterization of the non-adherentcells. (A) The number of non-adherent cellsproduced de novo in a 35 mm culture dish wasfollowed daily from D4, the onset of productionof floating cells, until the completion of theculture at D12. The number of floating cellsproduced remained at ∼2×104 daily from D4 toD8 and progressively increased to reach ameanof 1×105 at D12. (B) Flow cytometry analysis atD6 indicated that more than 90% of the floatingcells displayed strong QH1 immunostaining,indicative of their endo-hematopoieticphenotype. (C,D) Immunohistologicalcharacterization of the non-adherent cells byQH1-Alexa 488 staining (D) confirmed the flowcytometry analysis. (C) Phase contrast.(E-G) qPCR analysis of endothelial,hematopoietic commitment and hematopoieticdifferentiation-specific genes. The results areshown as the log-transformed fold change inexpression (PSM versus same stage embryo).(E) CD144 is strongly downregulated in all thefloating fractions tested, indicative of the loss ofendothelial-specific gene expression duringhematopoietic commitment and differentiation.(F) PU1 is upregulated from F4, the earliest timepoint of EHT, followed by an increase inexpression with time. (G) CD45 is upregulatedfrom F6 and increases with time. (H) The non-adherent cells were collected by cytospin andsubmitted to May–Grunwald Giemsa staining.Erythroblasts and differentiated granulocytesand monocytes were found to be abundant,indicative of multilineage differentiation. Errorbars indicate s.d.

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We then analyzed the role of serum in triggering ECcommitment. Since the cultures were supplemented with 5% FCSand 1% CS, we withdrew one or other serum and examined theeffect on the formation of flat cell layers and on AcLDL uptake byflow cytometry. Imaging and FACS analyses were performed at D4.Interestingly, withdrawal of FCS enhanced EC commitmentcompared with standard conditions, the percentage of AcLDL+

cells reaching 91.5±0.5% compared with a mean of 40±13% for thePSM in standard conditions (Fig. 5A-D,I). By contrast, removal ofCS resulted in a slight, non-significant decrease in the cellpopulation taking up AcLDL at D4 (Fig. 5E,F). However, CSabsence significantly impaired cell survival after D4, resulting insubstantial cell death at D6-D7 (not shown). As a baseline, we alsocompared somite tissue in the standard conditions (Fig. 5G,H).Taken together, this analysis revealed that the removal of FCS has adramatic effect on EC differentiation, strongly promoting the ECphenotype.Since VEGF and FGF are reported to be potent inducers of

angiogenesis and to synergize to promote vascular differentiation(Pepper et al., 1992; Asahara et al., 1995; Seghezzi et al., 1998), wefocused on these two factors by analyzing their influence on ECcommitment from the PSM.We removed one or other factor or both;we also removed all of the growth factors except VEGF. Amorphological analysis was performed at D1 and D4 (Fig. S3). Theabsence of VEGF caused a phenotype very similar to that when FCSis removed, with a robust emergence of flat, tightly adherent cells atD1 and the presence of very large areas of flat cells even at D3(Fig. S3C,D, compare with Fig. S3A,B). Removal of FGF, or bothVEGF and FGF, resulted in poor EC differentiation at D5 with novisible flat cell area (Fig. S3E-H). When VEGF was added as thesole growth factor, EC differentiation occurred but was readilyfollowed by the substantial production of round, hematopoietic cellsas soon as D2 (Fig. S3I,J).Finally, since the PSM generates several different cell types, we

questioned whether it is possible to direct the PSM cells towards thesmooth muscle cell lineage. TGFβ, which is known to promotesmooth muscle cell differentiation in culture, was added at 25 ng/mlto PSM cultures at D0 and replaced the initial set of growth factors.The layer of cells readily displayed a fibroblast-like morphology,with elongated cells forming a compact layer (Fig. 5J,K). Nobudding cell was found. Expression of FLK1, SCL, PU1 and CD45was absent from the culture, and CD31 was barely detected at D2and decreased thereafter (Fig. 5L), indicating a blockade in ECcommitment. Surprisingly, RUNX1 expression was still detectedbut was not associated with PU1 expression, one of its target genes.By contrast, alpha smooth muscle actin (αSMA) mRNA wasstrongly detected (Fig. 5L), suggesting firm commitment towardsthe smooth muscle cell lineage. These results demonstrated thephenotypic plasticity of the PSM culture according to the cultureconditions.

DISCUSSIONHere, using an easily amenable source of mesoderm, we report theestablishment and tuning of a versatile culture system using PSMcells that, with appropriate culture conditions, are able to faithfullyrecapitulate the cellular events that take place during the formationof the aorta and HSPC production. A notable feature is the biastowards the hemogenic endothelium lineage and the subsequentproduction of hematopoietic cells through EHT; another is the factthat a large number of cells undergo EHT, making it possible toeasily track cell fate changes at a single-cell level using time-lapsevideo microscopy.

The production of HSPCs from ECs was first shown in thechicken embryo in dye-marking studies (Jaffredo et al., 1998).Using interspecies grafting experiments, it was shown that HSPCsare produced at the expense of the hemogenic endothelialpopulation, which progressively disappeared from the aortic floor(Pouget et al., 2006). In mammalian embryos, phenotypic andgenetic approaches also demonstrated that the first HSPCs arederived from vascular ECs during a short period of time (de Bruijnet al., 2002; North et al., 2002; Zovein et al., 2008; Chen et al.,2009). Given the key role of the hemogenic endothelium and EHTin the production of HSPCs, an in-depth investigation of the cellularand molecular processes associated with these traits is needed.

Amajor hurdle in dissecting hemogenic endothelium commitmentand EHT is the low number of cells per embryo coupled with thedifficulties in isolating discrete steps associated with the cellularprogression. Significant advances have been made, using transgenicmouse lines carrying the +23 Runx1 hematopoietic enhancer (Swierset al., 2013), in understanding the molecular control of theendothelial-hematopoietic balance and the timing of these changes.However, hemogenic EC isolation remains difficult, especially if oneis to dissect discrete steps from non-hemogenic EC to EHT. Ourculture system allows 50-70% of the cells to be directed towards theendothelial and the hemogenic endothelial fates. This is an importantadvance comparedwith the numberof cells exhibiting these or similarphenotypes in ESC cultures (Eilken et al., 2009) or in embryos in vivo(Kissa and Herbomel, 2010; Yokomizo and Dzierzak, 2010). Inaddition, the stromal cell lines sometimes used in ESC differentiationprotocols into EC (Guo et al., 2007) or hemogenic EC (Eilken et al.,2009) are not required in our system, which facilitates cell isolation ifneeded. Another interesting feature is the fact that EHT andhematopoietic commitment occur without any modification to thecomposition of the medium. This indicates that the culture conditionsfaithfully recapitulate the molecular events occurring duringendothelial and hemogenic endothelial commitment in theembryonic aorta. Indeed, somitic ECs are not hemogenic, but it hasbeen shown that when these cells are placed in appropriate conditionsthey are able to give rise to hemogenic ECs and to blood (Pardanaudand Dieterlen-Lievre, 1999). In our case, we strongly bias the PSMcells towards the EC lineage and turn these non-hemogenic ECs intohemogenic ECs, as testified by the acquisition of endothelial traitsfollowed by RUNX1 expression during the first 4 days of culture.

Our daily molecular analysis indicates a progressive switchfrom the mesoderm to the hematopoietic state, with a passagethrough mesoderm, endothelium, hemogenic endothelium andhematopoietic fates. This is consistent with the proposed model ofblood cell formation deduced from ESC cultures (Lancrin et al.,2010).We found that the initial commitment ofmesodermal cells intoEC from D0 to D2 is not associated with RUNX1 expression.However, RUNX1 is expressed in culture fromD2, and its expressionincreases and extends to most, if not all, ECs thereafter. This is inkeeping with the changes in RUNX1 expression shown to occur invivo during the formation of the aorta (Richard et al., 2013), therebydemonstrating that our culture conditions accurately reproduce thecellular andmolecular events taking place in vivo.When they undergoEHT, each culture produces de novo at least 2×104 cells per 35-mmdish per day fromD4 toD8, and this number increases further to reach1×105 cells per dish at D12, corresponding to a considerablehematopoietic production considering the relatively low number ofcells seeded at culture onset. At themolecular level, qPCR analysis ofCD144, PU1 and CD45 reveals an early loss of endothelial traits infloating cells, in keeping with the changes that occur during EHT invivo, and a progressive increase in CD45 with time consistent with

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Fig. 5. EC differentiation depends on the tissue of origin and on medium composition. (A-I) Role of serum in EC differentiation. PSM in (A,B) standardconditions, (C,D) without FCS, (E,F) without CS, and (G,H) somites in standard conditions. Note the enhanced EC differentiation in the absence of FCS, with up to90% of the cells positive for AcLDL uptake, and the poor differentiation that is shown by the somite cells. Dot plots are from individual representative experimentsand are the result of 2000 events analyzed. (I) The percentage of AcLDL+ cells in the different experimental conditions. Data are mean±s.e.m. n=3. *P<0.01,**P<0.001, ***P<0.0001, Student’s t-test. Analyses are the result of three independent experiments with three independent wells per experiment.(J-L) Commitment to smoothmuscle cells with TGFβ. (J,K) Phenotype of the culture at D4 (J) and D8 (K) following replacement of the initial set of growth factors byTGFβ. No endothelial-specific phenotype was visible during the culture period (12 days). No hematopoietic-like floating cell was visible. The rare floating cells aredead cells. Boxed regions aremagnified in insets. Scale bars: 30 µm. (L) Semi-quantitative PCR analysis of the culture submitted to TGFβ. Cells were collected atD2, D4 and D6 and analyzed for the expression of several genes specific for endo-hematopoietic cells and for the alpha smoothmuscle actin isoform (αSMA) thatis specific for smooth muscle cells. Low to nil expression of endo-hematopoietic genes was found, whereas cells displayed increased expression of αSMA,indicative of smooth muscle cell commitment.

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maturation of the recently produced HCs (Jaffredo et al., 2005a; ZapeandZovein, 2011). This sustained hematopoietic production is indeeddue to the EHT that persists with time in culture, and also tohematopoietic cell multiplication. Future work will be needed toquantify the relative importance of these two events.The hematopoietic cells that are produced differentiate into several

hematopoietic lineages, as testified by the presence of at least threemorphologically distinct types of hematopoietic cell – granulocytes,monocytes and erythroblasts – originating from three distinct typesof progenitor. The lack of avian recombinant cytokines precludes athorough clonogenic identification of the progenitor cells producedin culture. Further work will be necessary to examine whetherlymphocytes, and eventually HSPCs, could also be produced. Thedifferential expression of PU1, which is a direct target of RUNX1(Huang et al., 2008), andCD45 betweenD4 andD6 indicates that thenewly formed HSPCs undergo progressive maturation in culture.Owing to the limitations mentioned above and the short duration

of the process, capturing hemogenic ECs undergoing EHT remainsa challenge in culture. A large number of cells experiencing EHT arevisible from D4. This unique situation would allow the isolationof hemogenic ECs undergoing EHT for analysis by variousapproaches. Investigation of the culture conditions has shown thatit is possible to reinforce EC differentiation or to anticipate EHT bymodulating the presence of FCS and VEGF. Our aim is to exploitthis versatility to find a way to collect sufficient numbers of cellsundergoing EHT for further cellular and molecular analyses.In addition to ECs and hemogenic ECs, we demonstrate that it is

possible to direct the culture towards the smooth muscle cell lineageby modulating the combination of growth factors. This opens theway to study, in greater depth, the molecular choices made bymesoderm cells during differentiation. This will also help to moreaccurately identify key factors involved in the EHT process fromfuture high-throughput data. Given the versatility of the culturesystem, one could also envision its use to manipulate the PSM cellsto produce striated muscle or tendon precursors, two cell types alsoderived following differentiation of the somite.Taken together, our results provide a new and versatile system

with which to study commitment to hemogenic ECs and the EHT.Future in-depth analysis of the molecular pathways involved inthese processes will have important implications for theunderstanding of EHT and the search for key hematopoieticinducing signals andmolecular pathways that are crucial in directingthe production of HSPCs from the hemogenic endothelium.

MATERIALS AND METHODSPSM isolationWe used quail (Coturnix coturnix japonica) embryo PSM, handled accordingto Fig. 1A. Eggs were incubated for 36-45 h at 37±1°C in a humidifiedatmosphere to reach 10-18 somite pairs. Microsurgery was performed aspreviously described (Pardanaud et al.,1996). The PSM was removed over alength corresponding to ten somites from both sides of the embryo. Fiveembryos (i.e. tenPSMs)were usedper culture dish. EachPSMwas cut into fiveor six equal pieces and rinsed in Opti-MEM plus GlutaMAX I containing 5%fetal calf serum (FCS), 100 units/ml penicillin/streptomycin and 1% chickenserum (CS) (all Gibco Life Technologies) before culture. Avian embryo careand procedures were in accordance with national and European laws.

Culture conditionsPSM was cultured in Opti-MEM with GlutaMAX I supplemented with 5%FCS, 1% chicken serum, 100 units/ml penicillin/streptomycin and thefollowing growth factors (PromoCell/PromoKine unless stated otherwise):human VEGF (C64410; 2 ng/ml), human FGF (C60240; 4 ng/ml), humanIGF (C60840; 3 ng/ml), human EGF (C60170; 10 ng/ml), hydrocortisone

(Sigma,H6909; 200 ng/ml) and ascorbic acid (Sigma,A4544; 75 μg/ml). Topromote smoothmuscle cell differentiation, the growth factors were replacedby TGFβ (PromoCell/PromoKine, C63500; 25 ng/ml). PSMwas cultured inCorning BioCoat 35 mm collagen I- or IV-coated dishes (Corning-Dutscher). Medium was changed every 2 days unless otherwise specified.

RNA extraction and qRT-PCRRNA extractions were performed using the RNeasy Kit (Qiagen). Freshlyisolated PSM (ten) were resuspended in the RNeasy buffer solution (RLT).For RNA extraction from cultured cells, the cells were first centrifuged(300 g for 10 min) to remove the medium and resuspended in RLT. Adherentcells were first trypsinized, washed in PBS containing 10% FCS,centrifuged and then resuspended in RLT. Quality and quantity of theextracted RNA was evaluated using Nanodrop. The primers used forsemi-quantitative RT-PCR and for qPCR are listed in Table S1. PCRwas performed on an Eppendorf Mastercycler Epgradient S. qPCRwas performed using the LightCycler 480 (Roche) real-time systemaccording to the manufacturer’s instructions. Relative expression wascalculated as 2[Ct(gene of interest)–Ct(gene of reference)].

Immunostaining and DAPI staining of in vitro cultureWe used acetylated low-density lipoprotein from human plasma coupled toAlexa Fluor 488 (AcLDL-A488; 1 mg/ml; Life Technologies, L23380,batches 1291485 and 1696210), the QH1 monoclonal antibody developedby Pardanaud et al. (1987) (obtained from the Developmental StudiesHybridoma Bank, created by the NICHD of the NIH and maintained at TheUniversity of Iowa, Department of Biology, Iowa City, IA 52242, USA) andantibody against RUNX1 (Abcam, ab92336, batches GR107772-3 and107772-5) to determine the endothelial phenotype of PSM-derived cells.AcLDL-A488 (1/100 in PBS) was incubated together with QH1 antibody(1/20 in PBS) for 30 min at 37°C in the culture dish.

After three washes in PBS, goat anti-mouse IgG1 secondary antibodycoupled to Alexa Fluor 555 (Molecular Probes; 1/100 in PBS), whichrecognizes QH1 antibody, was incubated for 30 min at 37°C. Cultures werethen washed three times in PBS before imaging.

When RUNX1 was revealed, the cells were fixed following AcLDLuptake with 3.7% formaldehyde in PBS, rinsed three times with PBScontaining 0.1% Triton X-100 for 10 min, incubated with the anti-RUNX1antibody for 1 h at room temperature, followed by incubation with a goatanti-rabbit secondary antibody coupled to biotin (Southern Biotech, 4050-08) followed by three rinses in PBS (5 min each) and an incubation withStreptavidin-Cy3 (Invitrogen, 43-8315) diluted 1/500 in PBS.

To visualize nuclei, cultures were first fixed in 4% paraformaldehyde inPBS for 20 min, washed three times in PBS, and then incubated with DAPIin PBS containing 0.4% Triton X-100 for 20 min. After three washes inPBS, cultures were mounted with a coverslip before imaging on a LeicaDM6000 B inverted microscope.

FACS analysisCells were stained with either AcLDL-A488 or with the QH1 monoclonalantibody. When adherent cells were used, cells were trypsinized and rinsedthree times in PBS. When floating cells were used, cells were gentlyremoved from the culture dish by pipetting. For AcLDL analysis, the cellswere suspended in PBS containing 7-aminoactinomycin (7AAD) to excludedead cells and analyzed with a MacsQuant analyzer 10 (Miltenyi Biotec).For QH1, cells were centrifuged, resuspended in PBS, incubated with theQH1 antibody (1/20) for 20 min at 4°C, washed in PBS and centrifuged.Cells were then incubated with goat anti-mouse IgG1-A488 (MolecularProbes; 1/100 in PBS) for 20 min at 4°C, washed in PBS and centrifuged.Finally, cells were resuspended in PBS containing 7AAD to exclude deadcells and analyzed on a FACSAria III (BD Biosciences) or on a MacsQuantanalyzer 10. Analyses were performed with FlowJo 10. Statistics wereperformed with GraphPad Prism.

May–Grunwald Giemsa stainingFloating cells were gently removed from the culture dish by pipetting,washed, centrifuged and suspended in PBS before proceeding to cytospin(Cytocentrifuge, Shandon-Elliot). Glass slides with the spot of cells were

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covered with May–Grünwald solution (Merck) for 3 min at roomtemperature. Five or six drops of PBS were added to the May–Grünwaldsolution directly on the slide, mixed gently, and incubated for another 3 min.Slides were then rinsed with PBS and covered with diluted Giemsa (1/10;Merck) for 20 min. Slides were finally washed with distilled water and airdried. Slides were mounted with a coverslip and a few drops of Entellan(Sigma). Images were taken on a Nikon Eclipse E800 microscope.

EHT trackingMovies were recorded on a Leica DM6000 B inverted microscope at 37°Cand 5%CO2 with a 10× objective. Images were acquired every 10 min usinga CoolSnap HQ2 camera (1392×1040 imaging pixels; Photometrics) over amean period of 24 h using Leica MMAF software v1.6.0. Films wereanalyzed using ImageJ (NIH). The tracking was made in two steps becauseof the two cell morphologies. In the first step, and because before undergoingEHT there is little movement in the flat layer of ECs, the cell undergoingphenotypic changes is marked at the time or immediately after the time (userchoice) of EHT. A circle is drawn to localize the cell based on its Cartesiancoordinates. From this point, a second window is opened that will allow theuser to trace the same cell back in time to before EHT (the number of framesback is also a user choice). Themacro is available upon request. In the secondstep, the algorithm automatically finds the brightest points around theprevious cell localization and selects the closest coordinates. Movies havebeen assembled and labeled using Final Cut Pro (Apple).

AcknowledgementsWe thank Drs Charles Durand and Cecile Drevon for critical reading of themanuscript; Laurence Petit for efficient help in flow cytometry; and Sophie Gournetfor excellent photographic and drawing assistance.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsL.Y., R.G. and T.J. designed experiments. L.Y., R.G., H.K., S.M. andM.S. performedexperiments. J.-F.G. developed the cell-tracking program. L.Y., R.G., H.K. and T.J.analyzed data. L.Y. and T.J. wrote the paper.

FundingThis study was supported by grants from the Fondation pour la Recherche Medicale[DEQ20100318258] and Agence Nationale pour la Recherche/California Institutefor Regenerative Medicine [ANR/CIRM 0001-02].

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.126714/-/DC1

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